Writing and erasing O-GlcNAc on target proteins in the brain

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WRITING AND ERASING O-GLCNAC ON TARGET PROTEINS IN THE BRAIN PROJECT SUMMARY O-Linked N-acetyl glucosamine (O-GlcNAc) is a nutrient sensor that dynamically modifies nuclear, cytoplasmic, and mitochondrial proteins. Dysregulation of O-GlcNAc has been linked to disruptions in sleep and circadian rhythm and several neurodegenerative diseases, including Alzheimer’s Disease (AD). While sleep and circadian rhythm defects are distressing symptoms of AD and other tauopathies, sleep disturbance may be a major risk factor for AD and is thought to accelerate its pathology. Extensive studies on the association of O-GlcNAc to AD have led to the first clinical trials targeting O-GlcNAc for therapy. However, these inhibitors globally alter the O- GlcNAc proteome, where a more targeted strategy may provide greater benefit. A systematic investigation of the connection between the O-GlcNAc modification and sleep regulation and AD pathogenesis would significantly impact the discovery of novel mechanisms to provide new avenues for targeted prevention and therapy. O-GlcNAc is regulated by nutrient availability and the complementary activity of two enzymes: O-GlcNAc transferase (OGT) writes the modification and O-GlcNAcase (OGA) erases it from proteins. Recently, innovations in protein engineering and gene editing tools developed by the co-investigators have provided access to precise tuning of O-GlcNAc on specific neurons and desired target proteins in the brain of Drosophila model systems of sleep and AD. Here, we will capitalize on the joint expertise in the Woo Lab and Walker Lab to facilitate the first systematic study to measure, map, and manipulate O-GlcNAc from desired target proteins and in specific neurons in the brain to yield crucial insights to the pathogenesis of AD and novel chemical strategies for remediation. To meet this goal, we will take a three-pronged approach. We will first systematically examine the relationship between O-GlcNAc in specific neurons of Drosophila models of sleep and AD pathogenesis to identify the brain regions that are most dependent on O-GlcNAc regulation. Second, we will use a targeted writer and eraser of O-GlcNAc, developed through protein engineering, to systematically examine the role of O-GlcNAc on selected target proteins in the brain to identify drivers and potential targets for alleviating sleep disruptions and AD pathogenesis. Third, we will pursue the discovery of small molecules that selectively write and erase O-GlcNAc in vitro and in vivo, which will complement our protein engineering approaches and provide targeted alternatives to global inhibitors that are under evaluation for AD therapy in the clinic. The successful outcome of this proposal will afford validated Drosophila models of sleep and AD with neuron-specific manipulation of OGT and OGA or specific target proteins using target writers and erasers of O-GlcNAc, with associated maps of O-GlcNAc proteins and sites, in addition to new and selective small molecule editors of O- GlcNAc to enable more targeted therapeutic approaches in the long-term. Additionally, the systematic methods and tools to connect physiological measurements to molecular function developed here will be translatable to the study of the connection of O-GlcNAc to other neurodegenerative diseases and beyond.